Ghost Membranes Research Articles

Catalysis of carbonic anhydrase IX and pH control in MDA‐MB‐231 breast cancer cells

Carbonic anhydrase IX (CAIX) is a membrane‐bound glycoprotein whose expression is considered a marker of tumor hypoxia and associated with tumor acidification. Our goal was to determine if the catalytic properties of native CAIX in MDA‐MB‐231 breast cancer cells are equivalent to those previously described in vitro and to evaluate its catalytic role in pH control. CAIX, like other CA family members, catalyzes the reversible hydration of carbon dioxide: CO2 + H2O ↔ H+ + HCO3−. We directly analyzed CAIX activity by using 18O exchange between CO2 and water as measured by membrane inlet mass spectrometry (MIMS). Data from membrane ghosts showed that the kinetic constants of CAIX catalysis in the membrane environment are very similar to those measured for purified, recombinant, truncated forms, suggesting that CAIX is not affected by the proteoglycan domain or the membrane environment. In intact cells, hypoxia increases CAIX activity compared to normoxia which is associated with increased expression. In the dehydration direction, the catalytic activity of CAIX increases as pH is decreased from pH 9 to pH 6.5. This suggests that at the pH of the tumor microenvironment (pH 6.8), CAIX uses protons to generate CO2. Thus, CAIX may contribute to both the development and maintenance of the acidic microenvironment of hypoxic tumors and thus to the survival fitness of tumor cells. BC073020 (SCF), DK45035 (SCF), and GM25254 (DNS)

Oxidative stress-dependent oligomeric status of erythrocyte peroxiredoxin II (PrxII) during storage under standard blood banking conditions

Although biochemical properties of 2-Cys peroxiredoxins have been extensively studied in various cell lines and organisms, redox-induced structural transitions of peroxiredoxin II (PrxII) in human erythrocytes certainly warrant further investigation. In this work, cytosol and membrane ghosts of both fresh erythrocytes (cells obtained just after blood collection) and 28-day stored erythrocytes were analyzed by proteomics tools. We demonstrated that in fresh red blood cells PrxII exhibits four different oligomeric states in cytosol, whereas no PrxII complexes are in the membrane. The highest molecular weight PrxII protein complex (440 kDa) was proven to derive from the association between tetrameric catalase (CAT, 232 kDa) and decameric PrxII, whereas oligomers at 140, 100 and 67 kDa resulted to be homo-polymeric complexes composed of variable copies of PrxII monomeric subunits. Interestingly, the 440 kDa complex contained both reduced and oxidized (disulphide-linked dimers) PrxII decamers. Upon oxidative stress (28-day storage), the PrxII oligomers at 100 kDa in the cytosol disappeared and the CAT–PrxII hetero-oligomeric complex at 440 kDa is converted to a higher molecular weight structure (480 kDa) due to the presence therein of cross-linked species of PrxII and hemoglobin. More interestingly, oxidized red cell membranes contained the CAT–PrxII complex detected in 0-day cytosol as a consequence of protein recruitments induced by oxidative stress, however it showed a greater percentage of PrxII dimers. Finally, since the adoption of distinct PrxII structures is known to be closely related to different functions, peroxidase activity assays were performed demonstrating a positive reaction for oligomers at 440 kDa (both in cytosol and membrane compartment) and at 140 kDa. Our results contribute to clarify structural and functional switching of peroxiredoxin II in erythrocytes, thus possibly opening new scenarios in the biological roles played by this protein in defense mechanisms against oxidative stress, especially with the reference to red cell storage lesions.

Erythrocyte membrane protein analysis by sodium dodecyl sulphate-capillary gel electrophoresis in the diagnosis of hereditary spherocytosis

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is currently the reference method for detecting protein deficiencies related to hereditary spherocytosis. The aim of the study was to evaluate an automated capillary gel electrophoresis system, the Experion instrument from BioRad, for its ability to separate and quantify the erythrocyte membrane proteins. The major erythrocyte membrane proteins (actin, protein 4.2, protein 4.1, band 3, ankyrin, α- and β-spectrin) were extracted and purified from membrane ghosts by centrifugation, immunoprecipitation and electroelution. Analyses were performed using SDS-PAGE and sodium dodecyl sulphate capillary gel electrophoresis (SDS-CGE) to establish a separation profile of the total ghosts. Then, the samples from patients received for investigations of erythrocyte membrane defects were analysed. Five of the seven expected erythrocyte membrane proteins were finally separated and identified. In the 20 studied cases, taking into account the screening test results and the clinical and family histories, the SDS-CGE method allowed us to achieve the same conclusion as with SDS-PAGE, except for the patient with elliptocytosis. The new SDS-CGE method presents interesting features that could make this instrument a powerful diagnostic tool for detection of erythrocyte membrane protein abnormalities, and can be proposed as an automated alternative method to the labour intensive SDS-PAGE analysis.

Green tea polyphenol epigallocatechin-3-gallate (EGCG) induced intermolecular cross-linking of membrane proteins

Increasing evidence has demonstrated that EGCG possesses prooxidant potential in biological systems, including modifying proteins, breaking DNA strands and inducing the generation of reactive oxygen species. In the present study, the prooxidant effect of EGCG on erythrocyte membranes was investigated. SDS–PAGE and NBT-staining assay were utilized to detect the catechol-protein adducts that generated upon treating the membranes with EGCG. The results indicated that EGCG was able to bind covalently to sulfhydryl groups of membrane proteins, leading to the formation of protein aggregates with intermolecular cross-linking. We suggested that the catechol-quinone originated from the oxidation of EGCG acted as a cross-linker on which peptide chains were combined through thiol-S-alkylation at the C2- and C6-sites of the gallyl ring. EGC showed similar effects as EGCG on the ghost membranes, whereas ECG and EC did not, suggesting that a structure with a gallyl moiety is a prerequisite for a catechin to induce the aggregation of membrane proteins and to deplete membrane sulfhydryls. EDTA and ascorbic acid inhibited the EGCG-induced aggregation of membrane proteins by blocking the formation of catechol-quinone. The information of the present study may provide a fresh insight into the prooxidant effect and cytotoxicity of tea catechins.

Urban PM2 5 induces ROS generation and RBC damage in COPD patients

Particulate matters (PM) produce adverse effects on the respiratory system and cause COPD. These effects are thought to involve intrinsic generation of ROS which are present in ambient PM (transition metals and aromatic organic compounds). Here, we examined the chemical composition and ultra-microscopic structure of PM2.5. The effect of this PM was studied in red blood cell (RBC) membranes (ghosts) from healthy volunteers (n = 11) and COPD patients (n = 43). These effects were compared with that produced by a Fenton metal-catalytic ROS generator. Oxidative biomarkers and cell damage were singificantly increased in presence of PM2.5 or ROS generator in RBC of COPD patients as compared with those in cells from healthy volunteers. In contrast, total SH groups, band 3 phospho-tyrosine phosphatase (PTPase) and glucose-6 phosphate dehydrogenase (G6PD) activities were all diminished in cells from COPD patients. In conclusion, PM2.5 increases damage to RBCs from COPD patients, decreases the activity of PTPase and G6PD, and alters the function of the anionic exchanger (AE1) and the antioxidant response by decreasing SH groups.

S-Nitrosylation of Rap1 and Relationship to Rap1 Activity and Disease Status In SCD

AbstractAbstract 2662Red cell adhesion appears to be a critical contributor to vaso-occlusion in sickle cell disease (SCD). We have been interested in intra-erythrocytic signaling events that can affect sickle red cell (SS RBC) adhesion and thereby stimulate vaso-occlusion. One such agonist-inducible signaling pathway is that of the β2 adrenergic receptor. β2 adrenergic receptor signaling pathways, involving both protein kinase A and the small guanosine triphosphatase Rap1, have been shown to affect multiple RBC adhesion receptors. Therefore, we sought to determine whether specific post-translational modifications of RBC membrane and intracellular proteins were associated with the signaling pathways affecting adhesion. Protein S-nitrosylation, a post-translational modification of cysteine thiol by nitric oxide (NO), is one of several post-translational protein modifications that can regulate diverse cell signaling pathways. Given the many reports of abnormal NO biology in SCD, we investigated how S-nitrosylation of erythrocyte proteins affects molecules involved in regulation of SS RBC adhesion. First, we identified RBC membrane proteins that underwent S-nitrosylation in RBCs from healthy donors (n=2) and SCD patients (n=6; three patients treated with hydroxyurea [HU] and three without HU treatment). Highly purified RBC membrane ghosts were pretreated with or without 0.05 mM CysNO (to promote S-nitrosocysteine formation) for 10 min, and S-nitrosylated proteins were isolated using the resin-assisted capture (SNO-RAC) method (Forrester et al. 2009). Captured S-nitrosylated proteins were visualized by SDS-PAGE, and protein identification was accomplished via an in-situ digestion of the bead-bound proteins, release of the residual bead-bound peptides, and analysis using nanoscale capillary liquid chromatography coupled to high resolution, accurate mass tandem mass spectrometry (LC/MS/MS). A number of proteins underwent S-nitrosylation in both normal and SS RBCs. Among these, MS analysis indicated that Rap1, which has previously been shown to affect SS RBC adhesion to laminin, underwent S-nitrosylation much more extensively in SS than in normal RBCs. These data were confirmed by SNO-RAC followed by immunoblotting. Using a RalGDS Ras-binding domain pull-down assay, we further quantitated the amount of detectable activated Rap1 in highly purified preparations of normal and SS RBCs (lacking either contaminating leukocytes or platelets), before and after RBC loading with NO. Active Rap1 was undetectable or minimally present in RBCs from healthy donors (n=6) but easily detectable in all SS RBCs (n=4). Loading of RBCs with NO in these same samples led to an increased amount of detectable activated Rap1 in both normal and SS RBCs. Finally, we found that patients admitted for pain episodes (n=3) had higher levels of Rap1 nitrosylation and activity than patients in steady state (n=5). In summary, we have found that Rap1, a protein that acts downstream of adrenergic signaling in RBCs, undergoes S-nitrosylation and that this modification is associated with increased Rap1 activity. Most importantly, SS RBCs contain significantly more S-nitrosylated Rap1 and activated Rap1 than do normal RBCs, and this difference in content of active Rap1 is more pronounced in patients with ongoing vaso-occlusion. Disclosures:Telen:GlycoMimetics: Consultancy.

An Improved Method for the Separation and Quantification of Major Phospholipid Classes by LC-ELSD

An improved HPLC procedure for the separation of phospholipids is described. The method described utilizes a solvent mixture of acetonitrile-methanol–water-trifluoroacetic acid (100:25:1.7:2.5, v/v) as the mobile phase, which is more compatible with the pump than mobile phases containing inorganic acids. Separation was by isocratic elution on a Hypersil silica column coupled to an evaporative light scattering detector. Complete separation of phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin (SM) was achieved in less than 20 min. The detection limits for PS, PE, PC and SM were 50, 50, 80 and 150 ng (S/N = 3), respectively. Human, bovine and porcine erythrocyte ghost membranes and animal tissues have been successfully analyzed for their phospholipid contents.

One-step direct reconstitution of biomembranes onto cationic organic polymer bead supports

In this study, we addressed the straightforward reconstitution of red blood cell (RBC) membranes on the surface of cationic organic polymer beads. The RBC membrane–bead complex was obtained by the incubation of white, unsealed rat RBC ghost membranes with a nonporous quaternary ammonium-type anion-exchange polymer bead with a 350–550 μm diameter. Confocal microscopic observations using a fluorescence membrane probe revealed that the RBC membranes were reconstituted on the outer surface of the bead without any remarkable structural gaps in the membrane. The absence of activity of two peripheral enzymes that latently reside on the cytoplasmic face of the RBC membranes demonstrated that the orientation of the RBC membranes immobilized on the beads was asymmetric as well as that in the native state. The RBC membrane–polymer bead complex was incubated with a primary antibody that is directed against the amino-terminal extracellular domain of the integral protein glycophorin A (GPA). The resulting complex was further incubated with a fluorescent secondary antibody and then subjected to confocal microscopic observations. Fluorescence resulting in the binding of the secondary antibody was found on the surface of the complex, which indicates that the amino-terminal extracellular domain of GPA is exposed to the surface of the complex. In addition, the anion uptake function of the most abundant integral protein anion-exchanger 1 (AE1) immobilized on the polymer beads was inhibited by pretreatment with its specific inhibitor 4,4′-diisothiocyano-2,2′-stilbene disulfonate, as is observed for the intact RBCs. Based on all these results, the RBC membranes were thought to be reconstituted on the ionic polymer beads by our one-pot procedure while maintaining the orientation and functions of the membrane proteins to some extent.

Antigen Specificity of the Humoral Immune Response to Mycoplasma haemofelis Infection

The aim of the present study was to characterize the antigenic specificity of the humoral immune response made by cats infected with the feline hemoplasma, Mycoplasma haemofelis. A crude M. haemofelis antigen preparation was prepared from red blood cells (RBCs) collected from a cat at the time of a high level of bacteremia. Plasma samples were collected from six cats before and after experimental infection with M. haemofelis, with regular sampling being performed from 15 to 149 or 153 days postinfection (dpi). Preinfection RBC membrane ghosts were prepared from these six cats and used to identify erythrocyte proteins that may have contaminated the M. haemofelis antigen preparation. The M. haemofelis antigen preparation comprised 11 protein bands. The immunodominant bands on Western blotting with infected cat plasma had molecular masses of 78, 68, 60, 48, and 38 kDa. Most cats (n = 5) had plasma antibody that reacted with at least one band (always including the one of 68 kDa) at 15 dpi, and all cats were seroreactive by 29 dpi. The maximum number of antibodies from an individual animal specific for an antigen was identified in plasma collected from 57 to 99 dpi. Contamination of the M. haemofelis antigen preparation with RBC membrane proteins was observed. The contaminating RBC proteins had molecular masses of from 71 to 72 kDa (consistent with band 4.2) and 261 and 238 kDa (consistent with spectrin), and these were recognized by all plasma samples. A range of M. haemofelis antigens is recognized by cats infected experimentally with the organism. These represent possible targets for immunoassays, but care must be taken to prevent false-positive results due to host protein contamination.

Spin Label Studies of the Hemoglobin–Membrane Interaction During Sickle Hemoglobin Polymerization

An enhanced hemoglobin–membrane association has been previously documented in sickle cell anemia. However, it is not known how this interaction is modified during the hemoglobin S polymerization process. In this work, we use a model of reconstituted erythrocytes from ghost membranes whose cytoskeleton proteins had been previously labeled with the 4-maleimido Tempo spin label, and that were subsequently resealed with hemoglobin S or A solutions. Using electron paramagnetic resonance spectroscopy, we studied the time dependence of the spectral W/S parameter, indicative of the conformational state of cytoskeleton proteins (mainly spectrin) under spontaneous deoxygenation, with the aim of detecting the eventual effects due to hemoglobin S polymerization. The differences observed in the temporal behavior of W/S in erythrocytes reconstituted with both hemoglobins were considered as experimental evidence of an increment in hemoglobin S–membrane interaction as a result of the polymerization process of hemoglobin S under spontaneous deoxygenation.

Determining radical penetration into membranes using ESR splitting constants

Determination of the depth of radical penetration into a lipid membrane is critical to the understanding of the role membranes play in radical attack. We have previously studied radical penetration into lipid bilayers using novel lipophilic spin traps and a combination of NMR and ESR techniques. We now focus on erythrocyte ghost (EG) membranes. Based on a correlation between ESR β-H splitting constants (a β-H) and solvent polarity, we have been able to locate stable radicals such as doxyls 2–4 and spin adducts 6–8 intercalated within liposomal bilayers and EG membranes. As a rule, the more lipophilic a spin adduct, the deeper it is found in the bilayer; however, the depth of penetration also depends on the steric bulk of the intercalant and whether intercalation is effected by sonication or diffusion, with the former more energetic and more effective. Compared to simple liposomes, the head group region of the red blood cell membrane is more rigid and lipophilic because of the presence of cholesterol. Hence, the biomembrane head group filters out possible intercalants that are not sufficiently lipophilic. Steric bulk plays less of a role in the EG system, perhaps because the cholesterol introduces a greater element of disorder, attenuating the role played by lipid–lipid interactions.

Interactions of therapeutically active plant flavonols with biological targets : Insights from fluorescence spectroscopic studies

Plant flavonols have attracted much recent attention in view of their novel therapeutic properties (effective against various free radical mediated and other diseases) which make them promising alternatives to conventional therapeutic drugs. However, till date, not much is known, regarding their mode of interactions and binding affinities with relevant biological targets. This article presents perspectives highlighting the usefulness of the exquisitely sensitive 'two color' fluorescence behavior of flavonols (which arise due to highly efficient photoinduced excited state intramolecular proton transfer (ESIPT) reactions) for exploring their interactions, at the molecular level, with biomembranes and proteins, which are the principal biological targets of such drug molecules. In this context, we made exploratory studies on the interactions of some representative therapeutically important flavonols with model and natural membranes (composed of phosphatidylcholine liposomes and red blood cell ghost membranes respectively) and serum albumin proteins. Since the ESIPT process is highly sensitive to external hydrogen bonding perturbation effects, the relative contribution between the two colors is strongly modulated by the local environment of the fluorophore, with dramatic changes in the emission yield, energy, anisotropy (r), lifetime (τ) and related parameters of both ESIPT tautomer and normal fluorescence bands. This provides multiparametric fluorescence probing opportunities, revealing salient details about the nature and location of binding sites as well as quantitative estimates of partition coefficients/binding constants. This promising new approach may be expected to open up new avenues for the 'screening' of the most appropriate flavonoid derivatives, from among numerous structural variants found in nature, as well as the design of relevant synthetic derivatives with improved features.

The covalent modification of spectrin in red cell membranes by the lipid peroxidation product 4-hydroxy-2-nonenal

Spectrin strengthens the red cell membrane through its direct association with membrane lipids and through protein–protein interactions. Spectrin loss reduces the membrane stability and results in various types of hereditary spherocytosis. However, less is known about acquired spectrin damage. Here, we showed that α- and β-spectrin in human red cells are the primary targets of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) by immunoblotting and mass spectrometry analyses. The level of HNE adducts in spectrin (particularly α-spectrin) and several other membrane proteins was increased following the HNE treatment of red cell membrane ghosts prepared in the absence of MgATP. In contrast, ghost preparation in the presence of MgATP reduced HNE adduct formation, with preferential β-spectrin modification and increased cross-linking of the HNE-modified spectrins. Exposure of intact red cells to HNE resulted in selective HNE–spectrin adduct formation with a similar preponderance of HNE–β-spectrin modifications. These findings indicate that HNE adduction occurs preferentially in spectrin at the interface between the skeletal proteins and lipid bilayer in red cells and suggest that HNE–spectrin adduct aggregation results in the extrusion of damaged spectrin and membrane lipids under physiological and disease conditions.

Blood Lipids in Antarctic and in Temperate-Water Fish Species

Antarctic fish live in very cold water and have adapted to this exceptional environment. Hemoglobin is absent or very low; yet these fish still have erythrocytes, and from these we prepared ghost-like membranes. We studied for the first time the lipid composition of ghost membranes and of plasma in Antarctic fish (C. hamatus and T. bernacchii) and compared our results with those obtained for temperate-water fish (C. auratus and A. anguilla taken from Lake Trasimeno, Perugia, Italy). The membranes of Antarctic fish were richer in glycerophospholipid (especially phosphatidylethanolamine), whereas the membranes of temperate-water fish were richer in sphingomyelin. Unsaturated fatty acids were particularly abundant in Antarctic fish: C. hamatus had long-chain unsaturated fatty acid (especially C22:6 omega-3), whereas T. bernacchii had shorter unsaturated fatty acyl chains (c16:1, omega-7). On the other hand, C. auratus and A. anguilla were particularly rich in C16:0, which constituted more than one-half of the total fatty acid. Plasma lipids (both phospholipid and cholesterol) were much more abundant in temperate-water fish. The differences in phospholipid content were mainly due to choline glycerolipids. Measures of membrane fluidity inferred from the fluorescence anisotropy of DPH indicated that the membranes from Antarctic fish were more fluid at any measured temperature than those obtained from fish living in temperate waters. The ability to live in a very cold environment has therefore been achieved by the two Antarctic species tested in this paper by different strategies, but with the same results on fluidity.

ATPase and acetylcholinesterase activities in erythrocyte membranes after incubation with glucose and in streptozotocin diabetic rats.

Enzyme activities of ATPases and acetylcholinesterase from isolated erythrocyte membranes (ghosts) were investigated before and after incubation with 50 mM glucose. Glucose incubation caused a time dependent loss of ATPase and acetylcholinesterase activities. Ghost enzyme activities in steptozotocin diabetic rats were found only insignificantly diminished.

Thiol-Dependent Membrane Transport of Selenium through an Integral Protein of the Red Blood Cell Membrane

The molecular details of the selenium metabolism and transport in living systems are still not completely understood, despite their physiological importance. Specifically, little is known about the membrane transport of selenium from most of the selenium containing compounds. In the present study, we investigated the mechanism for the membrane transport of selenium from red blood cells (RBCs) to the blood plasma. When the selenium distribution in the RBC ghost membrane after treatment with selenious acid was analyzed, nearly 70% of the selenium in the membrane was found to bind to the anion exchanger 1 (AE1) protein, which suggested that the integral protein AE1 is responsible for the membrane transport of selenium. The thiol dependency of the selenium export from the RBC to the blood plasma was examined using membrane permeable thiol reagents, i.e., N-ethylmaleimide (NEM) and tetrathionate (TTN). Treatment of the RBC with NEM, a thiol-alkylating reagent, resulted in modification of the thiol groups in the amino-terminal cytoplasmic domain (N-CPD) of the AE1, but not those in the membrane domain. Such an NEM treatment provided a marked inhibition of the selenium export from the RBC to the blood plasma. In addition, the treatment with TTN, a thiol-oxidizing reagent that forms intermolecular disulfide bonds, appeared to oxidize thiol groups in both the N-CPD and the membrane domain of AE1, which resulted in complete inhibition of the selenium export even during the initial period in which the export had a maximum velocity when using the thiol reagent-free treatment. Such complete inhibition of the selenium export from the TTN-treated RBC appeared to be due to the oligomerized AE1 proteins resulting from the intermolecularly formed disulfide bonds. These inhibitory effects using NEM and TTN suggested that thiol groups in the integral protein AE1 play essential roles in the membrane transport of the selenium from the RBCs to the blood plasma.

Cellular Membrane Disruption by Amyloid Fibrils Involved Intermolecular Disulfide Cross-Linking

Accumulating evidence has strongly suggested that amyloid fibrils of protein or peptide are cytotoxic. Fibrillar species appear to lead to disruption of cell membrane structures and thereby cause cell death. In this study, human erythrocytes were used as an in vitro model to examine the disruptive effect of lysozyme fibrils on the plasma membrane. Both the protofibrils and mature fibrils induced hemolysis and aggregation of erythrocytes. Treating ghost membranes with the fibrils resulted in aggregation of membrane proteins through intermolecular disulfide cross-linking. LC-ESI-MS/MS and Western blotting analysis showed that lysozyme fragments were incorporated into the aggregates of ghost membrane proteins, which suggested that thio-disulfide exchange among lysozyme and membrane proteins was triggered when the fibrils interacted with erythrocyte membranes. Metal-ion chelators, radical scavengers, and antioxidants had no effect on the amyloid-induced disulfide cross-linking. The exposure of interior hydrophobic residues and the increased level of solvent-accessible disulfides in the lysozyme fibrils are thought to be involved in membrane disruption. These results may unveil a novel pathway for the cytotoxicity of amyloid fibrils.

TSH induces co‐localization of TSH receptor and Na/K‐ATPase in human erythrocytes

Thyroid stimulating hormone (TSH) binds to a specific TSH receptor (TSHR) which activates adenylate cyclase and increases cAMP levels in thyroidal cells. Recent studies have reported the presence of TSH receptor in several extra-thyroidal cell types, including erythrocytes. We have previously suggested that TSH is able to influence the erythrocyte Na/K-ATPase ouabain binding properties through a receptor mediated mechanism. The direct interaction of TSH receptor with the Na/K-pump and a functional role of TSHR in erythrocytes was not demonstrated. The interaction of TSH receptor with Na/K-pump and a TSHR functional role are not yet demonstrated in erythrocytes. In this study, we examined the interaction between the two receptors after TSH treatment using immunofluorescence coupled to confocal microscopy and a co-immunoprecipitation technique. The cAMP dependent signalling after TSH treatment was measured to verify TSHR functionality. We found that TSH receptor and Na/K-ATPase are localized on the membranes of both erythrocytes and erythrocyte ghosts; TSH receptor responds to TSH treatment by increasing intracellular cAMP levels from two to tenfold. In ghost membranes TSH treatment enhances up to three fold co-localization of TSHR with Na/K-ATPase and co-immunoprecipitation confirms their direct physical interaction. In conclusion our results are compatible with the existence, in erythrocytes, of a functional TSHR that interacts with Na/K-ATPase after TSH treatment, thus suggesting a novel cell signalling pathway, potentially active in local circulatory control.

Antioxidative activities of some peptides isolated from hydrolyzed potato protein extract

Three peptides ( 5A, 5C and 6C), purified from potato protein hydrolysate fractions, possessed antioxidative activities. Isolation and purification were carried out using gel permeation chromatography and successive reverse-phase HPLC. These three peptide fractions were sequenced and identified as Phe-Gly-Glu-Arg, Phe-Asp-Arg-Arg and Phe-Gly-Glu-Arg-Arg, respectively. The fractions 5A, 5C and 6C inhibited linoleic acid oxidation by 55.3%, 58.5% and 61.7% using β-carotene decolorization assay system, while the inhibition ratio was 32.1%, 93.0% and 93.4% in the ferric thiocyanate assay system, respectively. The peptide fractions 5A, 5C and 6C also repressed lipid oxidation by 24.2%, 14.7% and 26.4% in the erythrocyte membrane ghost assay system, respectively. Oral administration of 100 mg/kg of body weight of the chemically synthesized peptides, 5A, 5C and 6C to rats (male Wistar) 30 min prior to ethanol injection reduced ethanol-induced gastric mucosal damage by 67.9%, 57.0% and 60.3%, respectively. Conclusively, these peptides have shown real potent antioxidative activities and could further be investigated for potential use as food additives.

Ceramide-Enriched Membrane Domains in Red Blood Cells and the Mechanism of Sphingomyelinase-Induced Hot-Cold Hemolysis

Hot-cold hemolysis is the phenomenon whereby red blood cells, preincubated at 37 degrees C in the presence of certain agents, undergo rapid hemolysis when transferred to 4 degrees C. The mechanism of this phenomenon is not understood. PlcHR 2, a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, induces hot-cold hemolysis. We found that the sphingomyelinase, but not the phospholipase C activity, is essential for hot-cold hemolysis because the phenomenon occurs not only in human erythrocytes that contain both phosphatidylcholine (PC) and sphingomyelin (SM) but also in goat erythrocytes, which lack PC. Fluorescence microscopy observations confirm the formation of ceramide-enriched domains as a result of PlcHR 2 activity. After cooling down to 4 degrees C, the erythrocyte ghost membranes arising from hemolysis contain large, ceramide-rich domains. We suggest that formation of these rigid domains in the originally flexible cell makes it fragile, thus highly susceptible to hemolysis. We also interpret the slow hemolysis observed at 37 degrees C as a phenomenon of gradual release of aqueous contents, induced by the sphingomyelinase activity. These hypotheses are supported by the fact that ceramidase, which is known to facilitate slow hemolysis at 37 degrees C, actually hinders hot-cold hemolysis. Differential scanning calorimetry of erythrocyte membranes treated with PlcHR 2 demonstrates the presence of ceramide-rich domains that are rigid at 4 degrees C but fluid at 37 degrees C. Ceramidase treatment causes the disapperance of the calorimetric signal assigned to ceramide-rich domains. Finally, in liposomes composed of SM, PC, and cholesterol, which exhibit slow release of aqueous contents at 37 degrees C, addition of 10 mol % ceramide and transfer to 4 degrees C cause a large increase in the rate of solute efflux.